It is generally accepted that the driving force for active transport, oxidative phosphorylation, and energy dependent pyridine nucleotide transhydrogenation in Escherichia coli is the protonmotive foce (delta uH ion) which is composed of a membrane potential (delta psi, inside negative) and a pH gradient (delta pH, inside alkaline) according to the relationship delta uH plus equals delta psi - (2.3RT/F)delta pH. Little is known of the mechanism which couples the protonmotive force to these energy-linked functions and it is this mechanism that we propose to study genetically and biochemically. We have identified a new genetic locus in E. coli (designated eup), mutations in which result in the uncoupling of the protonmotive force from active transport and the energy dependent transhydrogenase. Proposed studies include the isolation of additional Eup mutants and a complemntation analysis to determine the number of polypeptides coded for by the eup locus. To facilitate biochemical studies amber, deletion, and temperature-conditional Eup mutants will be isolated. New classes of energy uncoupled mutants will be sought using a selection procedure designed to isolate mutants pleiotropically defective in active transport. To define the biochemical functions of the eup gene product(s), a systematic comparison of the effects of missense, amber, deletion, and temperatuue-conditional eup mutations on active transport, oxidative phosphorylation, and energy dependent transhydrogenase will be done. To identify the eup gene product(s) membranes from Eup amber mutants will be analyzed by one- and two-dimensional polyacrylamide gel electrophoresis. Finally studies aimed at reconstitution of energy-linked functions in Eup defective membranes will be undertaken. The reconstitution system will then be used as an assay in the purification of the eup gene product(s).